Scaffolds are artificial structures capable of supporting a three-dimensional tissue formation. Scaffolds are supposed to resemble the connective tissue in an extracellular matrix. Thus, scaffolds allow for cell attachment, migration and growing of cells and synthesis of extracellular matrix components and biological molecules specific to the tissue targeted for replacement. To achieve those objects, a scaffold ideally provides a high porosity and proper pore size, a high surface area, biodegradability, proper degradation rate to match the rate of neotissue formation and it should provide a sufficient mechanical integrity to maintain the predesigned tissue structure. A scaffold should also be nontoxic to the cells (i.e. biocompatible) and should positively interact with cells including enhanced cell adhesion, growth, migration, and differentiated function (Ma, P. X., May 2004, Materials Today, p. 30-40).
The control of pore size is an important aspect for tissue engineering scaffolds. It has been shown in numerous studies that different pore sizes have different interactions for different cell types. One example to illustrate is a study done on the response of three cell types (canine dermal fibroblasts, vascular smooth muscle cells, and microvascular epithelial cells) to porous poly(L-lactic acid) scaffolds with four pore size distribution (<38, 38-63, 63-106, and 106-150 μm) (Zeltinger, J., Sherwood, J. K. et al., 2001, Tissue Engineering, vol. 7, no. 5, p. 557-572). Vascular smooth muscle cells displayed higher cell proliferation and extracellular matrix synthesis for larger pore sizes (106-150 μm). While, endothelial cells show a preference for pores less than 38 μm, forming a connected multicellular lining.
In addition, it is known that a specific range of pore sizes is required for different tissue engineering applications. For effective cell growth and tissue regeneration, a pore size range of 380 to 405 μm is required for chondrocytes and osteoblasts, 186 to 200 μm for fibroblasts and 290 to 310 μm for new bone formation. Therefore, it is important for scaffold fabricating techniques to be able to create three-dimensional (3-D) scaffolds of a range of controlled macro and micro-porosities to allow customization for different tissue engineering applications.
The creation of such scaffolds, with complex geometry for tissue regeneration purposes, has been one of the major challenges in tissue engineering. Fabrication techniques must provide three levels of control: (a) macroscopic and composition (mm to cm), (b) size orientation and surface chemistry of pores and channels for tissue ingrowth (hundreds of μm) and (c) locally surface texture and porosity (10 μm) (Griffith, L. G., 2002, Ann. N.Y. Acad. Sci., vol. 961, p. 83-95).
Many scaffolds used as implants and for tissue engineering purposes are fabricated by conventional methods (i.e., particulate leaching, fiber bonding, solvent casting, membrane lamination, melt molding, gas forming and phase separation). However, these methods are limited in that they typically generate scaffolds with simple macro-architectures and homogeneous microstructures, i.e. the micro- and macro pore size, geometry and connectivity in different layers of the scaffold cannot be well controlled (Yang, S., Leong, K.-F. et al., 2002, Tissue Engineering, vol. 8, no. 1, p. 1-11). However, since scaffolds are supposed to replace, for example, structural tissue like bone, cartilage, skin and esophagus, which do not normally have a complete homogeneous structure but are made of different layers which are structured depending on their special function and cell species which are growing in these different layers, artificial scaffolds also need to provide such a complex structure and not only a structure which provides only one specific pore size or one specific texture.
Rapid prototyping (RP) is a recent technology based on the use of computer control in manufacturing. Rapid prototyping takes virtual three dimensional designs from computer aided design (CAD), transforms them into thin, virtual, horizontal two-dimensional cross-sections and then creates each cross-section in physical space, one layer after the next until the three-dimensional model is finished. These two-dimensional layers can be made and bonded to previous layers one by one sequentially (Liu, Q., Sui, G., Leu, M. C., 2002, Computers in Industry, vol. 48, p. 181-197).
However, commonly used RP methods such as Fused Deposition Modeling (FDM) and Selective Laser Sintering (SLS) require elevated processing temperatures and this limits their ability to process temperature-sensitive polymers and bioactive components.
Ang, T. H., Sultana, F. S. A. et al. (2002, Materials Science and Engineering C, vol. 20, p. 35-42) describe a rapid prototyping method comprising bioplotting. This method is carried out at room temperature, where a solution is dispensed into a medium and subsequent reaction causes the solution to solidify and retain its dispensed path. The hydrogel scaffold obtained was subsequently frozen and freeze dried. Macropores of 400 to 1000 μm can be created with this technique by spacing the dispensed solution path. However, surface microporosity was not observed at all, as the coagulation process usually forms a skin at the surface.
Yan, Y., Xiong, Z., et al. (2003, Material Letters, vol. 57, p. 2623-2628) describe a system termed multi-nozzle deposition manufacturing (MDM) which is used to fabricate tissue engineered scaffolds for bone. Tricalcium phosphate (TCP) particles have been added to PLLA in dioxane and dispensed in a low temperature environment of under 0° C. The scaffold was subsequently freeze dried to obtain an interconnected porous structure at room temperature. The micropore obtained is about 400 μm while the micropores are about 5 μm. The scaffolds obtained comprise micropores of random orientation.
U.S. Pat. No. 6,899,873 B2 describes the manufacture of scaffolds by casting polymer solution into molds which are then frozen and freeze dried. Freezing temperatures and solvents can be used to control porosity and diameter of micropores. The ability to orientate the direction of the micropores is also demonstrated by creating a temperature gradient during freezing. PLLA scaffolds of different porosities (pore sizes from 50 to 100 μm) can be obtained. However, for this method it will be difficult to create three dimensional scaffolds with customized macro features. This method does also not allow fabrication of scaffolds having different micropore orientations and sizes within a scaffold.
Since there is a demand for scaffolds having a complex micro- and macrostructure further methods are required to provide such scaffolds.